The Hs0294 human malignant melanoma cell line produces a monolayer mitogen that stimulates the serum free growth of low-density cultures of Hs0294 cells. This report describes the purification of that mitogen, termed MGSA for melanoma growth stimulatory activity, from serum-free conditioned medium from the Hs0294 cultures. MGSA has been purified from acetic acid extracts of lyophilized conditioned medium by gel filtration, reverse-phase high-pressure liquid chromatography (RP-HPLC), and preparative electrophoresis, resulting in a greater than 400,000-fold purification. MGSA bioactivity resides in acid- and heat-stable polypeptides of high and low molecular weight (24-28 kd and less than 14-16 kd). However, the majority of the activity is reproducibly associated with the approximately 16-kd moiety eluting from RP-HPLC at approximately 35% acetonitrile. Reduction with dithiothreitol or B-mercaptoethanol results in a loss of biological activity but does not convert the 24-28-kd moieties to the less than 14-16-kd forms of MGSA. 125I-MGSA that has been purified by preparative electrophoresis (16 kd) specifically binds to Hs0294 melanoma cells and retains 100% of the growth-stimulatory activity. The 16-kd MGSA stimulates the proliferation of Hs0294 cells at concentrations of 0.3-30 pM. The electrophoretic mobility of MGSA is also unaltered by the preparative electrophoresis procedure, further demonstrating that this procedure does not alter the biochemical integrity of the growth factor. Purified MGSA does not enable anchorage-independent growth of normal rat kidney (NRK) cells and is therefore different from the previously described transforming growth factors. The amino acid composition of MGSA differs from that of other previously described growth factors. These data demonstrate that MGSA represents a separate class of growth factors with biological and biochemical properties different from other growth factors.